Biomed & Pharmacother (1990) 44,69-83 0 Elsevier, Paris

69

ossier Pat~o~io~ogy of mye~odys~~~stic sy~d~Qrnes I Blazsek, G Math6

Summary - The myelodysplastic/preleukemic syndromes represent unique clinical situations since patients with initially mild hemopoietic abnormalities can be singled out from those progressing into frank myeloid leukemia. Here we confront data focused on the identification of critical cellular, molecular biological, cytogenetic and physiological defects leading to leukemic progression. An increasing amount of data supports our earlier hypothesis according to which the impairment of an endogenous (int~cellular} life-cycle suppressor gene-product, or functi~ally related regulatory genes. plays the decisive role in the course of disease progression. The identification of systemic as well as clonally tr~smissible defects have clinical importance since in some cases the therapeutic application of the appropriate physiological substances may result in long lasting hematological remission. myelodysplastic syndrome I pathobiology R&sum4- Pathobiologie des syndromes my~l~yspl~tiques.

Les syndrames my~lc~yspiasriq~fes~pr~lez~c~m~qrrPs rep&enayant in~tialemeFFtdes a~~~mali~s h~mapo~~tiq~~espeu graves pe~~~‘eflt vers lflle leffc@mio my&iofde franrhe. Dans reite revue. nous cnnfrontons les donndes pour tenter d’idontifer les d6fuu.r cytologiques, hiaiogiques malkwlaires. cytoghne’tiques et physinlngiques cnnduisant d la progression de la myilodysplasie vers la Ieur4mie. Les rtkrltats rkents ranfirmenr nntre hypathke selnn iaquelle l’inacli~*ation d’rn r&ulateur suppressifel endo@?ne du cycle cellulaire. ou de terrains g&es rtigulareurs. powaienr jouer Ie rale dkisif au caurs de la 1eucfimogenPse. L’identifcation des dgfaurs sw4miqnes etlou transmissibkes de ceNule ii cellule a un inttWt rlinique imparranr car dans certains ~~idrames, ~app~i~a~ion ih~rapel~iiqtle des subslances ph?sio~o~iq~~esappropri~es peut a~ai~tir & une r~~issio~l h~ma~ola~iq~~ede langue d&e. tei&l des ~ondi~~o~~s cli~~iq~fesuniques, car /es ~tieflts itre s&i&s de ceux qui progressent irr&ersihlemenr

syndromes my&odysplasiques I pathobiologie

Introduction The myelodysplastic syndromes (MDPS) cover a wide range of hemopathies characterized by a hypercellular bone marrow (BM) accompanied by a variety of genetic, phenotypical and functional abnormalities and by a paradoxical decline in mature blood cell production. Since the recognition by Dustin in 1949 that anemia may precede acute myeloid leukemia (AML) [29] and by HamiltonPeterson in 1944 that some anemicfpreleukemic patients ultimately transfo~ into frank AML 1471, our knowledge about MDPS has been considerably enriched. Despite more recent molecular biological findings, the progress in the treatment of MDPS patients is still very controversal. The

steadily increasing frequency of MDPS and its close relationship to frank AML justify the need for further efforts in understanding the pathobiology of this complex hemopathy. Here we confront clinical observations and experimental data suggesting the hypothesis that impairments along the metabolic pathway of some intracellular suppressor factors may underly the progression of MDPS.

Clinical a~~earan~e~ ~la§si~cat~on,life expectancy Historically, those suffering from MDPS have

been singled out from anemic patients on the

70

1 Blazsek, G Math6

basis of long lasting persistance of the disease

that could not be reverted by treatment with metabolic adducts. Indeed, erythroid abno~alities al.ways occw early following the onset of disease and a wide range of red blood cell defects have been described in MQPS: including metabolic [20] and morphological dysfunctions [59], reappearance of fetal hemoglobin [ 1031 and surface antigen alterations [69]. In a particular form of WPS, mitochondrial iron deposition occurs which is accompaned by erythroid “sideroblastic” hyperplasia. Peripheral thrombocytopenia may appear first in an isolated form. However, it generally characterizes the most severe forms of MDPS. A series of defects have been described varying from the production of micromegakaryocytes with no platelet granule formation to the extremely low level of functionally defective megakaryocytes. The myeloid cell lineages show several forms of differentiation defect. The granulocyte may appear with a hypo- or hypersegmented nucleus, the cytoplasmatic granulation is frequently defective which is accompanied by low peroxidase and/or paradoxically elevated (ri naphtyl acetate esterase activities. In contrast to other cell lineages, the frequency of mon~yte/macrophages is usually increased. In extreme forms this gives rise to one distinct clinical entity: chronic myelomono~ytiG leukemia (CMML). These anomalies may occur in various combinations and transitory episodes due Eo infections or conditions of stress, as well as long lasting changes from one subtype into another making the MDPS difficult to interpret. Since 60-85% of patients die following opportunistic infection or hemorrhage, extensive research has been carried out to identify preleukemic patients and to classify MDPS more precisely. Disease progression can be simply expressed by using the cumulation of the Boumemouth score which attributes one point to each of the following clinical parameters: C log/d1 hemoglobin, K 2.5 x lo3 granulocyte/pl blood, c 100 000 platelets, and > 5% BM blast WI]. The modified FAB system is widely accepted and more specific for the classification of the preleukemic stages [8]. It applies the BM blast percentage, the gradual increase of which reveals disease progression: a) less than 5% blast cells designates refractory anemia (RA), h) 520% blast cells, RA with excess blasts (RAEB), and C) 20-30% blast cells, RAEB in transfo~ation (RAEBT). RAEBT is crucial in the prediction of frank leukemic transformation and 2 5% peri-

pherai blast cells or the presence of Auer rods also designates this subtype. A recent publication on the tenement of RAEBT reveals the difficulty of finding any simple scoring system: patients who have less than 30% marrow blasts but higher than 30% peripheral blast cells can be separated from MDPS and be classified as having peripheral acute leukemia [ 191. Patients with chronic myelomonocytic leukemia (CMML) have absolute peripheral blood monocytosis (> log/L) often associated with granulocytosis. The primary acquired sideroblastic anemia (PASA) represents another distinct subgroup. In these latter 2 forms survival at one year is 2 95%. in contrast to RA and RAEB where the disease progression is more accelerated and life expectancy shortened to 64% and 35% respectively [60]. Cfonaf origin and progcniror cell anomalies in MDl’S

The “one cell, multiple hits” theory of carcinogenesis, proposed according to epidemiological data by Muller [82] and Nordling 1871 suggested that a critical effect of the first mutation might be to increase the growth fraction of the mutated cell [lOOI. If this criteria is satisfied in a suspected cell ~pulation, only one more duplicate genetic hit is required to fit the empiric mathematical model of cancer development. Indeed, in the 2 most freqnent myeloid leukemias, 2 stages can be clearly distinguished. In chronic myeloid leukemia (CML) the long-lasting chronic phase is characterized by the clonal dominance of the Phc population, which is ultimately followed by the emergence of more aggressively growing blast cells with multiple chromosomal defects. IDIacute myeloblastic leukemia (AML), despite the rapid disease progression, the 2 stages can be distinguished by analysis of restriction fragment length poly~rphism (RFLP). These studies showed that phenotypically normal diffe~ntiated blood cells (segmented neutrophil granulocytes), that reappear following intensive chemotherapy, belong to the same abnormal clone which has first occurred and which may reappear during disease progression as an aggressively growing, undiffcrentiatcd blast cell population [31]. A growing amount of pathophysiological and experimental data suggests that the MDPS does not in general fit well into the above model, while

the terminal phase shows some similarities with AML.

Pathobiology of myelodysplastic syndromes

The fact that a less aggressive form of MDPS always changes into another form and ultimately may transform into frank leukemia mirrors the course of disease progression where multiple alterations occur, being essential to the full expression of the leukemic phenotype. There is evidence to suggest the possible clonal stem cell origin of some MDPS. Genetic analysis of cellular mosaicism in a woman heterozygous for glucose6-phosphate dehydrogenase (G6PD) and exhibiting constitutive chromosomal abnormality showed not only the monoclonal stem cell origin of the hematolymphopoietic cell compartment but also demonstrated that a single chromosomal anomaly (47Xx+8 or 46XX de1 [ 111 (q23) in this study) is not sufficient for the full expression of the leukemic phenotype [102]. Cell kinetic data show that both erythroid and myeloid cell populations traverse the cell cycle in a disproportionate manner. It is important to note that more cells were residing at the Go/G1 boundary the higher the risk was of acute transformation [79, 951. Indeed, studies on clonogenic hemopoietic progenitors generally showed that decreased frequency and/or impaired growth potential both of the granulocyte-macrophage Colony Forming Cell (GM-CFC) [II, 110, 1141 and of erythroid Burst Forming Lrnit IBFLI! i72j correlated with disease progression. In patients, whose BM cells showed constitutive chromosomal abnormalities, the in vitro GMGFC population expressed the same chromosomal defects in 4 cases out of 6, indicating a cloneby-clone variation in colony forming ability in vitro [891. The poor progenitor cell growth and the longlasting ineffective blood cell production reveal a paradox in view of the notion of the monoclonal stem cell origin of MDPS. Available data cannot explain why the impaired functioning of 1 mutated stem cell not yet having aggressive growth potential would result in a significant and longlasting decrease of the total blood cell pool. It could be suggested that at least in RA and in PASA, normal stem cells are still functioning in sufficient numbers to maintain the correct blood cell level. This is, however, not the case, and we therefore suggest an alternative possibility that systemic impairment in regulatory factors that fundamentally determine the expansion, terminal differentiation and cellular egress from the BM may preceed the occurrence of the first clonal alteration.

71

immunological dysfunctions in MDPS Infectious episodes frequently underlie disease progression. Microbial infection is known to activate the peripheral immune system which in response produces a battery of cytokines. In model experiments the bacteriai LPS induces a burst of CSA production within a few hours [iO8]. However, this activation, which also includes a rise in cachectin/TNF, is followed by a very long-lasting period (up to 6 months) when the peripheral immune system becomes refractory to any further LPS stimuli [74, 771. Therefore, the pathopb;rsiological role of inhibitory cytokines in provoking aberrant terminal differentiation at the BM level cannot be excluded. Preclinical and clinical data provide indirect support for this notion since prednisone, which inhibits suppressor T-cell and some macrophage functions, was able to increase the in vitro growth of GM-CFC populations and it induced a clinical and hemopoietic remission in MDPS patients [4, 111. Data on fully transformed AML cells also support this notioi! providing evidence that in some cases leukemic myelobasts may constitutively produce TNF [90]. The role of the endogennus (autocrin) hemopoieric growth factor production in leukemogenesis The survival, clonal expansion and some of the end-cell functions of BM cells show an absolute dependence on polypeptide hemopoietic growth factors (HGFs) including Interleukin 3 (IL-3). IL6 and granulocyte and/or macrophage Colony Stimulating Factors (GM, G- and. M-CSF) [23,751 (table I). In addition, permanent myeloid cell lines and myeloid blast cells from primary AML patients also-show a requirement for one or more HGFs, therefore a positive role of HGFs in leukemic transformation has been postulated [27, 55.76, 1131. The immortalized but not tumorigenic murine hemopoietic cell line, FDC-PL, which is dependent on IL-3 or GM-CSF, when infected with the Abelson virus [24] or transfected by the bcr/abl construct [49] are transformed into autonomous leukemic cells, and they may produce IL4; GM-CSF or IL-3 [58]. However, the autocrin GM-CSF production is neither sufficient nor necessary to leukemic transformation. This has become evident from studies on transgenic mice expressing the GM-@SF gene, which resulted in massive monocytosis, blindness and death but not in frank leukemia [66]. In addition, the analysis of GM-CSF gene expression in AML blast cell

I Blazsek. G MathC

72

Table L Biological characteristicsof the principal hemopoietic cell growth factors [23,775.76.98). Name

Molecular

Acronym

weight (kDa)

Multipotential colony stimulating factor

Multi CSF (IL-3)

Gramdocyte-macrophageGM-CSF (CSFa) colony stimulating factors

CIwonfosonral k~~isatian~pu’

Target colony

Producer

forming cells

cells

IS-3woO

5q 23-3 I

n,e,bGC,Mo,E,Mk

T-cell

1%3OOoo

Sq 23-3 1

n,eGC.Mo,E,Mk

T-cell Endothelial cell Fibroblast

Granulocyte colony stimulating factor

G-CSF (CSFB)

2oOoo

17q 11.2-21

nGC

Monocyte Fibroblast

Macrophage colony stimulating factor

M-CSF

36-52000 70.9oOoo

5q 33.1

MO

MO

Monocyte Fibroblast Endothelial cell

Interleukin-6

IL-6

19-21000

7p I5

(IFN%MGll Erythropoietin

Epo

28000

7P

E(BFU.CFU-E)

Kidney. macrophage

Thmmbopoietin

TKV

?

?

Mk

Thrombopenic serum

populations clearly indicated that the autocrin HGF production is not common in AML Pl, 1181. In MDPS patients autocrin HGF production has not been demonstrated at the genetic level. BM cells from CMML patients were recently reported to produce in culture a significant amount of IL-6 and GM-CSF in 7 out of 7 and in 2 out of 7 cases respectively [30]. Earlier studies also demonstrated that elevated CSF level may frequently occur in MDPS patients [34]. The authors, however, do not provided direct evidence that the CFC and the HGF producing populations were identical, In conclusion, the physiological HGFs may play a positive role in the course of leukemic progression since they may support the survival of preleukemic cells. However, autocrin HGF production is not a common mechanism necessarily underlying the progression of leukemia. Indeed, more recent data suggest that there is no specific reason why autonomous cell growth would require the constitutive upregulation of growth factor gene(s) or their receptor(s). It becomes increasingly evident, that in all the eukaryotic cells studied, the progression of the cell cycle is determined at 2 critical points: 1) at the “restriction point” or “start” of the Gt phase upstream from which an interacting cascade of biochemical chain reactions lead to the complete

duplication of the genome, and 2) at the S/G2 phase boundary where a mitotic cycle inductor emerges assuring rapid cell division. Considerable comparative data have accumulated, from yeast to human cells, leading to the conclusion that both the restriction point and the mitotic division are instructed by evolutionary, highly conserved homologous proteins that can be interchanged between distant species. The restriction point is bypassed by the effect of proteins identical, or homologous to the “cell division cycle” protein (cdd) of fission yeast which is a 34kd protein kinase or to the Maturation Promoting Factor alfa [15, 671. The inductory cdc2 protein, shows at least 2 specific, cell-cycle phasedependent functions. It is suggested that through the Go/G1 phase, the dephosphorylated retinoblastoma gene product plOS/l IO-Rb inhibits cdc2 activation [17, 211. When plOS/llO-Rb is neutralized or phosphorylated, this leads to cdc2 activation and to a rapid cell-cycle progression. At the GdM phase, another mitotic inducer emerges which is named cyclin (or p63 cdcl3) [68, 991. Cdc2 and cyclin form a transient complex with serinethreonin histon 1 kinase activity which is believed to drive the mitotic processes [15, 991. It is interesting to note that cdc2 and cdcl3/cyclitt function during the early stage of embryonic

Pathobiology of myelodysplastic syndromes development [68] when rapid “prokaryot type” cell divisions occur through repeated S and M phases without Gr and G2 phases and before tissue specific growth regulators become dominant. This fact led to the suggestion that the critical biochemical hit that ultimately determines the neoplastic/leukemic transformation should concern the above intracellular cell cycle inductory proteins. It has been established that specific phosphorylation inactivates and dephosphorylation activates these cell-cycle regulatory proteins. Therefore, an important key role is attributed to

73

a family of protein kinases/phosphatases [73] that may mediate the extracellular growth factor signals to the nuclear cell-cycle regulators. In the hemopoietic system, the linkage between specific growth factors and the intracellular cell cycle inductors has not yet been established. However, available data now makes it possible to design a hypothetical model where exogenous cell lineage specific and intracellular/nuclear proteins can be clearly separated (fig 1).

abortive

complex

v-myc Large T (Papova virus SV40) E7 (Papilloma Virus) ElA (Adenovirus)

ApoFtosis (activ cell de;llh) Figure 1. Exogenous and intracellular regulaiXS of the mitotic cell cycle. A model of leukemogenesis. A model of mitotic cell-cycle. After cell division (M-mitosis/cytokinesis) a differentiated daughter cell (end-cell) may undergo active cell death (apoptosis) if adequate pleiotropic growth factor is not accessible. The progression of the interphase through the Gt phase is driven by specific hormonal factors (here GM-CSF). GM-CSF stimulates the global RNA and protein synthesis resulting in cell enlargement. At the Restriction Point an intracellular “cell division cycle” inductory protein arises (p34cdc2). Simultaneously the cell-cycle inhibitory Retinoblastoma gene product (plO5/1 IO Rb) is neutralized by protein kinase phosphorylation or by the formation of “abortive” protein complexes whereby the cdc2 induces a rapid S-phase progression. At the end of S-phase the level of another critical cell-cycle promoter the cdcl3/cyclin increases. cdcl3/cyclin forms a temporal complex with cdc2 which thus possesses histon HI kinase activity. At the end of M the cdcl3/cyclin degrades suddenly and shortly after the Rb protein may undergo dephosphorylation inhibiting the GI progression probably by forming a complex with free cdc2 [5. 15. 17. 21. 67. 6% 73, 7% 99 1.

74

I Blazsek. G Mathi

Constitutive chromosomal defects and oncqwe dysfunctions in MDPS

Although extracelhdar soluble mediators may promote the pregression of MDPS, much more emphasis has been placed upon genetic defects leading to the generation of preleukemiclteukemic cell populations. Interestingly, each of the dominant regulatory pathways of the cell cycle include some cellular oncogene product(s) suggesting thgt studies on oncogenes has opened a sufficiently large encugh window to approach the basic mechanisms of ce& cycle control and to understand the neoplastic ~nsfo~ation. The close linkage between MDPS and AML indicates that constitutive, duplicable defects of genomic functions characterizing the blastic cell populations in AML may arise at the decisive hit of preleukemia progression. Earlier studies have shown that 8ross chromosomal abnormalities were prcscnt ln 6O-70% of AML, patients (primary and secondary cases taken together) [lOa]. The high resolution banding technique now makes it possible to identify chromosomal defects in nearly all AML patients ~8~1~%) llt9]. Specific chromosomal defects were found in about 30% of cases. Not surprisingly many of them coincided with the genomic location of cellular oncogenes (table II). Characteristically, in those patients where specific chromosomal defects occurred, the corresponding oncogenes were significantly over expressed. These include c-myc (lo-30 x in AFL), c-myb (10 x ), c-ets 1 (10-30 x in AMMoL), c-H ras, c-ki-ras. c-fcls, c-festfps, C-SW, c-e&l3 [38]. In model experiments, constitutive overexpression of an exogenous c-my& gene has been shown to block di~crentiation in 2 murine e~th~leukemia cell lines [85] and antisense c-myb oligonucleotides that inhibit p7S*‘“* expression appeared to block the growth of hemopoietic progenitors [39]. However, despite the large number of oncogene defects identified in fresh AML blast cells or in model experiments there is little information on oncogene functions in MDPS. Molecular genetic studies revealed the possibility that the ras oncogene family (H-ras, Ki-ras, N-ras 1 encoding homologous membrane bound proteins of 2lkd which resemble the G-proteins with signal transducting function, is involved in le~kemogenesis. In MDPS, activation of the N-t-as gene by point mutation in codon 13 has been de-

tected by an in vivo selection assay in nude mice with transfected NIH 3T3 cells in about 40% (318) of cases [54]. Others have found that the mutation of N-ras was much less frequent and the K-ras oncogene may also be mutated: out of 34 patients studied, mutations at codon 12 of K-t-asoccurred in 1 out of 6 patients with RAEB and in 2 out of 9 cases with CMML. Surprisingly, no correlation was established between the presence of ras mutations and disease progression into AML [71]. Studies on the H-ras polymorphism of rare alleles which have been identified in 17% of AML cases has shown that the presence of rare alleles is not a common predisposing factor in MDPS [18]. These studies could not establish the com~n role of ras mutation in the progression of MDPS. Model experiments provided evidence that ras, like the src gene product 1161,may have dual effects. Microinjection of activated p2 1 ras (the protein product of human WHa-c-ras-oncogen) into PC12 (pheochromocytoma) cells induced cell differentiation [6]. The result has been confirmed by v-ras retrovirus infection. [86]. Concordant with these findings, in the ovarian granulosa cell model, co-transfection of granulosa cells with simian virus 40 and ffa-ras oncogen generated stable cell lines capable of induced steroidogenesis [l]. In conclusion, ras gene products that promote cell proliferation and induce neoplastic transformation in NIH 3T3 cells, have the opposite effect in other cell types: PC12 or granulosa cells. Clearly the exact role of ras mutations in the initiation and progression of MDPS still awaits further clarification. Classical chromosomal abnormalities are much less frequent in MDPS than in AML. Using the premature chromosome condensation (PCC) technique, however, which makes it possible to detect struct~~ralchromatin abno~alities in interphase cells 1611 Mittelman et al succeeded in demonstrating that the genomic organization is frequently impaired in MDPS [52]. A similar conclusion was reached from the analysis using the high resolution banding technique [ 1201. Among stable chromosomal defects, specific deletions or monosomies are of particular interest 1981.In preleukemia the -5/5q- and -7/7q- deletions appear most frequently, in 16% and 19% of patients respectively. Other characteristic anomalies affect chromoso~ lq, 2, t~somy 8,9q, llq, 15q, 16q 17q and 2Oq (table II).

Pathobiology of myelodysplastic syndromes Table II.

75

Genomic localizationof some critical genesinvolved in the extracellular and endogenous control of cell growth and

differentiation [32.35,38,39,46,48,54,71,76,98,106, Chromosome

Oncogen

107,119.120].

Hemopoietic growthfactor

Growth factor steroid hormone receptors

Tumor suppresor genes

__ _-

~11-13 .N-ras p32 .L-myc ~34-36 .C-src2

inv lq del lq t(l;ll)

~23-24 .N-myc

de12

~21-23 .erbA2p - .hap 934 .c.fms ---

6

7

.R-T3fi .RAR2 q14 q22-32 q233 1 q33

*IL1 .IIA,lLS

934 .R-M-CSF - .R-CC

.lL3,GM.CSP

~15 .1L6 q2l .Epo

-_ -_ __

___

-_ -_

mono 7 de1 7pl7q

---

-_ -_

trisomy 8

-_

del9q

924 .c-myc - .c-mos

-_ -_

9

q34 .c-abl

-_

11

~14.1 .c-Ha-ras q22-24 _---

---__ --

12

p 12.1 .c-Ki-ras

--

- .R-VD3

--__ -_ -_

-_ -_ -_ _-_

14 15 16

-_-_q21-31 .c-fos

20

ql l/12 .c-erbA -_ ql2/13 .csrcl

22

q13.1 .c-sis

17

pi3 pll -

q26.1 .cfes/fps ---

de1 5q15q

__ - .R-Eo __

8

I3

q13-I5 .Poliiis -_ __

.M-CSF

-pl l/l2 .cKi-ras q21-23 .c-myb pter22 .c-et&B -_

Kayotypic anomalies in MDPS

-_ __ q I I .2-2 I .G-CSF __q12 .LIF

- .R-GC -ql l/l2 .R-‘I% q21.1 .RARl

_-

.Wilm’s t. Rhabdosarc. .Bladder .Breast .Hepatosarc.

del llq t(l;ll) -

.Retinob. .Osteosarc. .Lung see .Breast .Bladder

-

-q14 -_

del l5q

---

t(16;17) dell6

p .Colorectal t. (tl6; 17) __ __

--

del2Oq

76

I Blazsek, G Math6

Apart from t~somy 8, all of the constitutive defects identified in MDPS patients represent to SOme extent the loss of chromatin material. This may be the cornerstone of the preleukemic/leukemic populations since in contrast to the generally accepted hypothesis it suggests that the congenital or acquired loss of some differentiation-inducing gene products, rather than the excess of endogenous HGF production, is responsible for the initiation of leukemogenesis. On the nature of a suggested leukemia suppressar mechanism In general terms the preleukemic/leukemic transformation can be explained as a result of constitutive, clonally duplicable defects in regulatory genes whose function is to suppress cell cycle prog~ssion thus pe~itting the full expression of a differentiated phenotype. The existence in differentiated end-cells of an endogenous differentiation cell cycle inhibitor (DCI) fulfilling the criteria of a cell-cycle suppressor gene-product has been suggested on a theoretical basis [5]. Cytological, molecular biological and genetic results have emerged recently supporting the hypothesis that endogenous tumor suppressor genes and gene products exist whose neutralization or deletion is central to neoplastic transformation 132, 37, 641. Although data are accumulating on suppressor genes in a number of solid tumors especially on the retinoblastoma susceptibility gene, Rb, [48, 1071, an analogous regulatory system has not been described to-date in the case of premalignant/malignant hemopathies. The polypeptide myeloid Leukemia Inhibitor Factor (LIF) was thought to have a similar function. LIF induces monocyte differentiation of the murine myeloid cell line Ml [Sl]. Simultaneous studies, however, elucidated that LIF/DIA inhibited the differentiation of normal embryonic stem cells 11091 and paradoxically it may even stimulate the growth of some permanent myeloid cell lines (DA) [801. Therefore, the biological function of LIF seems to be more complex than that of the tumor suppressor gene products. Independent data from different experimental approaches provide evidence that the lipophyl steroi~thyroid hormones, all trans Retinoic Acid (trRA) and la, 25dihydroxyvitamin D3 (1,25D3) may function in an intracellular regulatory network that controls morphogenesis, tissue specifi-

cation and terminal differentiation [94, 96, 97, 1171. These hormones control gene expression through highly conserved, homologous nuclear receptors. The hormone-activated receptor complex recognizes specific DNA sequences within the promoter of target genes via the “zinc-finger”, a common motif in a number of recently identified transcriptional regulators. Each member of this hormone family has been shown to modify hemopoietic cell production (table III). Interest has been focused on trRA and 125D3 since both appeared to have a regulatory effect on several genes involved in cell-cycle control and terminal differentiation f78]. The following arguments support their key role in the maintenance of EM homeostasis. Although quiescent normal hemopoietic progenitor cells do not respond to trRA or to 1,25D3 in the absence of polypeptide HGFs, these progenitor cells show a complicated pattern of growth and differentiation responses when adequate HGFs are provided, indicating synergistic interactions between the cell lineage specific HGFs and the rather ubiquitous steroid ho~ones 126, 841. Similarly to normal progenitor cells, permanent factor-independent hemopoietic cell lines also showed cell-lineage specific responses: a) very early undifferentiated cells (KG], KG la) were refractory to both trRA or 1,25D3, and b) more differentiated cell lines could be induced by I.253133 towards a monocytoid differentiation (HL-60, U 937) and by trRA towards granulocytic maturation (HL-60). Fresh BM blast cells from patients with MDPS could always be stimulated to growth and differentiate into macrophages with 1,25D3 [92], and trRA inhibited rather than stimulated GM-CFC in contrast to their normal counterparts [26, 841. Gross chromosomal defects the S/Sq-syndrome, t [16, 171 in MDPS, specific deletions in ANLL on chromosome 3q-, Sq-, and the t (15;17) in APL all suggest that the constitutive lack of a nuclear receptor for members of the steroid/thyroid hormone, trRA, 1,25D3 hormone family may fundamentally underlie leukemic transformation. The Sq- syndrome also shows that the endogenous auto&n production of IL-3, IL-4, IL-5 as well as GM and M-CSF do not contribute to leukemogenesis in these specific cases [98]. The translocation 15; 17 in APL provides a very important insight into the possible molecu-

Pathobiology of myelodysplastic syndromes

77

Table III. The effects of I ,25(OH)zD3 on gene expression in normal and transformedcell populations. Gene activation or suppression was assessed at the level of DNA (t) or RNA (m) [63,78,84,92,117]. Normal cells

Gene

Effect

Transformed cells

Gene

Effect

Hematopoietic

stem cells progenitor

? ?

MNC Monocyte

Ret TrFe Interleukin-1 Lysosyme Heat shock prot Diacylglycerol aryltransferase

T-Lymphocyte activated

B-lymphocyte

t

Fibronectin 63D3 ag.diff HLA-Dr c-fos

-l(m)

T t

c-fms(FtecM-CSF) TNFa PKC(Rec TPA) c-myc c-myb Histon H4

Interleukin-2 GM-CSF 60 y) the application of allogeneic BMT is very limited. In 3 series, however, using histocompatibility locus antigen (HLA)-matched donors on a total of 58 patients aged between 4 and 54 years, BMT has been judged successful in 22 cases [3, 7, 881. This indicates that for younger patients, if a compatible donor is available, aiiogenous BMT should be considered as a life saving treatment of MDPS. Despite the close relationship between MDPS and ANLL, clinical trials using high-dose

Pathobiology of myelodysplastic syndromes chemotherapy failed to reverse disease evolution [2]. More emphasis has been placed upon lowdose Ara-C regimens since Ara-C appeared to induce gene expression and differentiation in model experiments by interfering with DNA synthesis. Indeed, the administration of low doses of Ara C (10 mg/m’/d for up to 42 d [ 1011 or 20 mg/m*/d for 14-21 d [45] by continuous infusion gave marked improvements in the hematogrammes, indicating some differentiation-promoting effect of Ara C in viva However, the chromosomal abnormalities were not abrogated and the survival time was only slightly prolonged by low dose Ara-C. Perhaps the most surprising discovery which hallmarks these recent years of research is the rapid, long lasting therapeutic effect with minimal side effects, of the hormonally active all trans Retinoic Acid in patients with acute promyelocytic leukemia [25, 33, 571. This clinical result has importance in both fundamental and clinical research since it brings together precise molecular-biological, cytogenetic and cell-biological results with a human disease and perhaps provides a model for the elaboration of new treatment regimens. The use of Retinoic Acid in MDPS demonstrated that trRA may induce the regression of hemopoietic abnormalities [41]. More importantly, it significantly prolongs the median survival time rate as compared to age-matched non-treated patients [22]. Initial trials with 1.25cholecalciferol indicate that this secosteroid hormone may have beneficial effects on MDPS patients [104]. However, hypercalcemia is a major limitation of its prolonged application. Recently a number of vitamin D3 analogues have been developed with differentiation-inducing properties but without k viva hypercalcemic effects [63]. These new analogues have not yet been applied in MDPS. One of the most characteristic pathobiological features that distinguishes MDPS from ANLL is the poor growth potential in vitro and the long population doubling time in viva of the abnormal myeloid progenitor cells. Since the proliferation, differentiation and survival of normal progenitor cells show an absolute requirement of HGFs, and on the other hand, recombinant human GM-CSF and G-CSF have been shown to accelerate the engraftment and hematological recovery following BMT [46], the application of GM-CSF or G-CSF in MDPS with low BM blast count seemed to be rational. In a recent case report on therapy-related MDPS it has indeed been demonstrated that the administration at 60-120 pg rhGM -CSF!m2/d for

79

14 d significantly stimulated the non-clonal hematopoiesis and interestingly it down-graded ;he neoplastic clone for more than one year [ 1121 as judged by cytogenetic analysis. In other clinical trials with GM-CSF on primary MDPS patients, and by using the more powerful PCC techniques it has been revealed that despite the increased WBC count upon rhGM-CSF administration for 15 d the ratio of abnormal to normal cells remained constant [53]. In another series, a rapid rise of BM blast percentage was revealed (up to 60%) indicating the risk that GM-CSF may even accelerate disease progression [36]. However, it is important to mention here that in a recent trial on ANLL, the combination of the GM-CSF with high dose chemotherapy, in sequence, resulted in a long lasting hematological remission suggesting that a critical fraction of leukemic progenitor cells that could escape during conventional chemotherapy, became accessible to the cytotoxic drug on the effect of GM-CSF (Takaku, 1989, personal communication).

Conclusion During the past few years important progress has been made in understanding the pathobiology of MDPS. Recent trials with physiological substances gave promising results even when they were applied as monotherapy. Hopefully, future efforts using combinations of growth and differentiation inductors, new microbial agents (bestatin) and analogs of physiological agents (heme-arginate [ 1151). non hypercalcemic vitamin D3 analogues [63] will further improve the efficacy of antileukemic regimens.

Acknowledgments The authors wish to thank M L Labat and J Br6ard for critical discussion, N Vriz and MJ Ferreira for preparing and C Hamilton for rereading this manuscript. and I Moshe for preparing the artwork. A part of this work was supported by ARC and ACB.

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